Ripk1

Following cell transfection, total protein was extracted, separated on SDS/PAGE and blotted onto nitrocellulose membranes (Bio‐Rad, Hercules, CA, USA). Membranes were probed with specific primary antibodies [ZNF611 (Abcam, Cambridge, MA, USA); ZNF304 (Abcam); RIPK1 (BD Transduction Laboratories, Lexington, KT, USA); DUSP8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); HRAS (Abcam); TNFR21 (Santa Cruz); and β‐actin (Cell Signaling Technology, Beverly, MA, USA)], followed by horseradish peroxidase‐conjugated secondary antibodies anti‐mouse (Zymed Laboratories Inc., San Francisco, CA, USA) or anti‐rabbit (Zymed). Immunodetection was by chemiluminescence (Super Signal^® West Femto; Thermo Scientific, Waltham, MA, USA). Densitometry analysis was performed using imagej, version 1.45 (National Institute of Health, Bethesda, MD, USA).

All chemicals and reagents used were of analytical grade and were purchased from Sigma Aldrich (St. Louis, MO, USA). Annexin V-FITC apoptosis detection kit and 7-aminoactinomycin D (7-AAD) were received from Biolegend (San Diego, CA, USA). Antibodies for immunoblotting including PARP (Santa Cruz, sc-25780), cPARP (Cell Signaling, D64E10), laminA/C (Abcam, ab133269), caspase-8 (MBL, M032-3), caspase-10 (MBL, M059-3), caspase-9 (MBL, M054-3), caspase-3 (Cell signaling, 9665), anti-DR4 (Abcam, ab8415), anti-DR5 (Abcam, ab8416), GAPDH (Santa Cruz, sc-47724), HSC70 (Santa Cruz, sc-7298), BiP-1 (Cell signaling, C50B12), IRE1 (Cell signaling, 14C10), RIPK1 (BD Bioscience, 610459), XIAP (Santa Cruz, sc-55550), and survivin (Abcam, EP2880Y). Bradford reagent was obtained from Bio-Rad (Marnes-la-Coquette, France). Chemiluminescent Western blots detection kit was purchased from Advansta (San Jose, CA, USA).

Pierce Co-Immunoprecipitation Kit (Thermo Fisher Scientific, USA) was used to detect interaction of RIPK1 and RIPK3 in accordance with the guidelines. Briefly, the cells with indicated treatment were harvested and lysed by IP Lysis Buffer. The primary antibody RIPK1 (BD Biosciences, USA) was added into the lysis solution containing the total protein, followed by using Sodium cyanoborohydride, Coupling Buffer, Elution Buffer and Loading Buffer, to obtain the experimental samples. RIPK1 was used to the control of IP, and the expression of RIPK1 and RIPK3 was detected by western blot analysis.

Whole cell lysates were prepared with TNE lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% v/v NP-40, 3 mM EDTA, supplemented with Complete protease inhibitor mixture (Roche)). The protein lysates were separated by SDS-PAGE, transferred onto nitrocellulose membranes and reactive proteins were detected with antibodies for RIPK1 (BD Biosciences), RIPK3 (Abnova, Heidelberg, Germany), MLKL or actin (Sigma-Aldrich) via chemiluminescence (Lumiglo, Cell Signaling, Danvers, MA, USA). RIPK3 expression was quantified using the program ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA). To compare expression levels of RIPK1 and RIPK3 in tumor cell lines, identical amounts of protein (20 μg) were loaded, using lysates from PancTu-I cells as a standard on each gel, and identical exposure times were taken to allow a direct comparison of expression levels. For the quantitative analysis of the relationship between the levels of RIPK3 and the specific sensitivity of the respective tumor cell line to TRAIL/zVAD/CHX-induced programmed necrosis, values for RIPK3 expression were normalized between the gels and calculated relative to CCRF-CEM cells. In all Western blots, detection of actin served as a loading control.